RÉSUMÉ
Current effective therapies for autoimmune diseases rely on systemic immunomodulation that broadly affects all T and/or B cell responses. An ideal therapeutic approach would combine autoantigen-specific targeting of both T and B cell effector functions, including efficient removal of pathogenic autoantibodies. Albeit multiple strategies to induce T cell tolerance in an autoantigen-specific manner have been proposed, therapeutic removal of autoantibodies remains a significant challenge. Here, we devised an approach to target both autoantigen-specific T cells and autoantibodies by producing a central nervous system (CNS) autoantigen myelin oligodendrocyte glycoprotein (MOG)-Fc fusion protein. We demonstrate that MOG-Fc fusion protein has significantly higher bioavailability than monomeric MOG and is efficient in clearing anti-MOG autoantibodies from circulation. We also show that MOG-Fc promotes T cell tolerance and protects mice from MOG-induced autoimmune encephalomyelitis. This multipronged targeting approach may be therapeutically advantageous in the treatment of autoimmunity.
Sujet(s)
Autoanticorps , Encéphalomyélite auto-immune expérimentale , Souris , Animaux , Lymphocytes T , Glycoprotéine associée à la myéline , Encéphalomyélite auto-immune expérimentale/anatomopathologie , Glycoprotéine MOG/toxicité , AutoantigènesRÉSUMÉ
Significant progress in elucidating the genetic etiology of anxiety and depression has been made during the last decade through a combination of human and animal studies. In this study, we aimed to discover genetic loci linked with anxiety as well as depression in order to reveal new candidate genes. Therefore, we initially tested the behavioral sensitivity of 543 F2 animals derived from an intercross of C57BL/6J and C3H/HeJ mice in paradigms for anxiety and depression. Next, all animals were genotyped with 269 microsatellite markers with a mean distance of 5.56 cM. Finally, a Quantitative Trait Loci (QTL) analysis was carried out, followed by selection of candidate genes. The QTL analysis revealed several new QTL on chromosome 5 with a common core interval of 19 Mb. We further narrowed this interval by comparative genomics to a region of 15 Mb. A database search and gene prioritization revealed Enoph1 as the most significant candidate gene on the prioritization list for anxiety and also for depression fulfilling our selection criteria. The Enoph1 gene, which is involved in polyamine biosynthesis, is differently expressed in parental strains, which have different brain spermidine levels and show distinct anxiety and depression-related phenotype. Our result suggests a significant role in polyamines in anxiety and depression-related behaviors.
Sujet(s)
Anxiété/génétique , Dépression/génétique , Complexes multienzymatiques/génétique , Phosphoric monoester hydrolases/génétique , Stress psychologique/génétique , Animaux , Anxiété/métabolisme , Anxiété/physiopathologie , Chromosomes de mammifère/génétique , Dépression/métabolisme , Dépression/physiopathologie , Femelle , Mâle , Souris , Souris de lignée C3H , Souris de lignée C57BL , Complexes multienzymatiques/métabolisme , Phénotype , Phosphoric monoester hydrolases/métabolisme , Polymorphisme de nucléotide simple , Locus de caractère quantitatif , Spécificité d'espèce , Spermidine/métabolisme , Stress psychologique/métabolisme , Stress psychologique/physiopathologieRÉSUMÉ
Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the (15) N isotope effect on Escherichia coli cultures that were grown in either unlabeled ((14) N) or (15) N-labeled media by LC-ESI-MS/MS-based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between (14) N and (15) N-labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between (14) N and (15) N-labeled bacteria. Our data demonstrate for the first time that the introduction of the (15) N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.
Sujet(s)
Escherichia coli/métabolisme , Marquage isotopique/méthodes , Isotopes de l'azote/composition chimique , Protéomique/méthodes , Chromatographie en phase liquide , Neutrons , Protéome/analyse , Protéome/composition chimique , Protéome/métabolisme , Spectrométrie de masse en tandemRÉSUMÉ
Recent findings in the literature suggest a relation between histidine triad nucleotide-binding protein-1 (HINT1) and psychiatric disorders such as major depression, anxiety, and schizophrenia, although its physiological roles are not completely comprehended. Using Western blot, we compared HINT1 protein expression in the postmortem dorsolateral prefrontal cortex and thalamus of schizophrenia patients and healthy controls for contributing to elucidate the role of HINT1 in schizophrenia pathophysiology. HINT1 was found to be downregulated in the dorsolateral prefrontal cortex and upregulated in the thalamus. Our results combined to previous studies in human samples and preclinical models support the notion that HINT1 must be more explored as a potential target for psychiatric disorders.
Sujet(s)
Régulation de l'expression des gènes/physiologie , Protéines de tissu nerveux/métabolisme , Cortex préfrontal/métabolisme , Schizophrénie/anatomopathologie , Thalamus/métabolisme , Adulte , Sujet âgé , Sujet âgé de 80 ans ou plus , Femelle , Humains , Mâle , Adulte d'âge moyen , Statistique non paramétriqueRÉSUMÉ
Several reports have implicated a role for the histidine triad nucleotide-binding protein-1 (Hint1) in psychiatric disorders. We have studied the emotional behaviour of male Hint1 knockout (Hint1 KO) mice in a battery of tests and performed biochemical analyses on brain tissue. The behavioural analysis revealed that Hint1 KO mice exhibit an increased emotionality phenotype compared to wildtype (WT) mice, while no significant differences in locomotion or general exploratory activity were noted. In the elevated plus-maze (EPM) test, the Hint1 KO animals entered the open arms of the apparatus less often than WT littermates. Similarly, in the dark-light box test, Hint1 KO mice spent less time in the lit compartment and the number of entries were reduced, which further confirmed an increased anxiety-related behaviour. Moreover, the Hint1 KO animals showed significantly more struggling and less floating behaviour in the forced swim test (FST), indicating an increased emotional arousal in aversive situations. Hint1 is known as a protein kinase C (PKC) interacting protein. Western blot analysis showed that PKCγ expression was elevated in Hint1 KO compared to WT mice. Interestingly, PKCγ mRNA levels of the two groups did not show a significant difference, implying a post-transcriptional PKCγ regulation. In addition, PKC enzymatic activity was increased in Hint1 KO compared to WT mice. In summary, our results indicate a role for Hint1 and PKCγ in modulating anxiety-related and stress-coping behaviour in mice.
Sujet(s)
Anxiété/génétique , Anxiété/physiopathologie , Comportement animal/physiologie , Protéines de tissu nerveux/métabolisme , Animaux , Corticostérone/sang , Adaptation à l'obscurité/génétique , Modèles animaux de maladie humaine , Comportement d'exploration/physiologie , Régulation de l'expression des gènes/génétique , Mâle , Apprentissage du labyrinthe/physiologie , Souris , Souris knockout , Protéines de tissu nerveux/déficit , Protéine kinase C/métabolisme , ARN messager/métabolisme , Statistique non paramétrique , Natation/psychologieRÉSUMÉ
Brain proteome analysis of mice selectively bred for either high or low anxiety-related behavior revealed quantitative and qualitative protein expression differences. The enzyme glyoxalase-I was consistently expressed to a higher extent in low anxiety as compared with high anxiety mice in several brain areas. The same phenotype-dependent difference was also found in red blood cells with normal and cross-mated animals showing intermediate expression profiles of glyoxalase-I. Another protein that showed a different mobility during two-dimensional gel electrophoresis was identified as enolase phosphatase. The presence of both protein markers in red or white blood cells, respectively, creates the opportunity to screen for their expression in clinical blood specimens from patients suffering from anxiety.
Sujet(s)
Anxiété/métabolisme , Protéines du sang/métabolisme , Modèles animaux de maladie humaine , Amygdale (système limbique)/composition chimique , Amygdale (système limbique)/métabolisme , Animaux , Anxiété/induit chimiquement , Anxiété/diagnostic , Comportement animal , Marqueurs biologiques/analyse , Marqueurs biologiques/métabolisme , Protéines du sang/analyse , Technique de Western , Électrophorèse bidimensionnelle sur gel , Femelle , Humains , Lactoyl glutathione lyase/métabolisme , Mâle , Souris , Enolase/métabolismeRÉSUMÉ
Secreted protein, acidic, and rich in cysteine (SPARC) is thought to regulate cell matrix interaction during wound repair. We hypothesized that SPARC might promote migration via integrin-dependent mechanisms. The present study was designed to clarify the contribution of SPARC in the wound healing process after myocardial infarction (MI). Adult mice received a specific alpha(v) integrin inhibitor or vehicle through osmotic mini pumps. Mice of each group were either sham-operated or MI was induced. SPARC expression was investigated 2 days, 7 days, and 1 month after the surgical procedure. For migration assays, a modified Boyden chamber assay was used. A transient increase of SPARC levels was observed, starting at day 2 (2.55+/-0.21), day 7 (3.72+/-0.28), and 1 month (1.9+/-0.16) after MI. After 2 months, SPARC expression dropped back to normal levels compared to sham-operated hearts. Immunofluorescence analysis showed an increase of SPARC in the infarcted area 2 days after MI, a strong increase in the scar area 7 days after MI, and only low levels in the scar area 2 months after MI. Integrin alpha(v) inhibition abolished the up-regulation of SPARC. In vitro migration assays demonstrated that fibronectin-stimulated haptotaxis of fibroblasts was modulated by SPARC. This study provides evidence that SPARC is significantly up-regulated in the infarcted region after MI. This up-regulation is dependent on alpha(v) integrins. As SPARC is found to regulate fibroblast migration, it appears to play an important role in the injured myocardium with regard to healing and scar formation.
Sujet(s)
Fibroblastes/anatomopathologie , Infarctus du myocarde/anatomopathologie , Ostéonectine/métabolisme , Animaux , Mouvement cellulaire , Cellules cultivées , Protéines de la matrice extracellulaire/génétique , Protéines de la matrice extracellulaire/métabolisme , Femelle , Fibroblastes/métabolisme , Fibronectines/métabolisme , Régulation de l'expression des gènes , Intégrines alpha/métabolisme , Intégrines/métabolisme , Mâle , Souris , Souris de lignée C57BL , Infarctus du myocarde/métabolisme , Myocytes cardiaques/effets des médicaments et des substances chimiques , Myocytes cardiaques/métabolisme , Myocytes cardiaques/anatomopathologie , Ostéonectine/effets des médicaments et des substances chimiques , Ostéonectine/génétique , Facteur de croissance dérivé des plaquettes/pharmacologie , Facteur de croissance transformant bêta/pharmacologie , Facteur de croissance transformant bêta-1 , Vitronectine/pharmacologieRÉSUMÉ
Aberrant migration of smooth muscle cells (SMCs) is a key feature of restenosis. Since extracellular matrix proteins and their receptors of the integrin family play a critical role in this process, it is instrumental to understand their contribution to cell migration and invasive motility of SMC on the molecular level. Therefore, we investigated the role of alpha(v)-containing integrins expressed by primary human coronary artery smooth muscle cells (hCASMCs) in vitronectin (VN)-initiated signaling events and cell migration. In hCASMC plated on VN, alpha(v)-containing integrins were localized at focal adhesion sites. Haptotactic stimulation through VN led to a dose-dependent increase in cell migration and concomitantly to enhanced tyrosine phosphorylation of focal adhesion kinase. Both events were completely blocked by a specific inhibitor of integrin alpha(v). Additionally, the integrin alpha(v) inhibitor abolished PDGF-BB-stimulated chemotactic migration. Confocal microscopy confirmed the increased tyrosine phosphorylation at VN-initiated focal contact sites in hCASMC, that was abolished upon alpha(v) inhibition. In vitro invasion of hCASMC was severely compromised in the presence of the integrin alpha(v) inhibitor paralleled by decreased levels of secreted matrix metalloprotease 2 (MMP-2). Together, integrin alpha(v) inhibition abrogates tyrosine phosphorylation at focal adhesion sites and diminishes MMP-2 secretion leading to reduced migration and invasion of hCASMCs.